Novel clock gene promoter

ABSTRACT

The present invention discloses a Period2 gene promoter, a construct containing the promoter and a reporter gene, a cell containing the construct, a transgenic animal harboring the construct, and a method for screening a substance which controls expression or oscillatory expression of a biological clock gene. The screening method uses the above cell, a suprachiasmatic nucleus section or peripheral tissue of the above transgenic animal or the above transgenic animal.

TECHNICAL FIELD

The present invention relates to a biological clock gene promoter, aconstruct containing the promoter and a reporter gene, a cell containingthe construct, a transgenic animal harboring the construct, and a methodfor screening a substance which controls expression and/or oscillatoryexpression of a biological clock gene.

BACKGROUND OF THE INVENTION

It is known that a large number of organisms are possessed of amechanism in their bodies, which generates circadian rhythm with ablout24 hour cycle (Reference 1). In mammals, the mechanism generatingcircadian rhythm controls sleep-wake rhythm, blood pressure, bodytemperature and a part of hormone secretion rhythm (Reference 2,Reference 3). As diseases caused by the circadian rhythm disturbance,sleep-wake rhythm disorder (delayed sleep phase syndrome (DSPS), non-24hour sleep-wake syndrome), seasonal depression, jet lag syndrome(JET-LAG), sleep disturbance in night and day shift workers, nocturnalporiomania and delirium found in patients with senile dementia and thelike have been reported (References 4 to 7). In addition, there is areport stating that a part of children with school refusal or workerswith refusal to attend firm as a social problem is caused by a circadianrhythm disorder (Reference 8). Increase in the number of patients withrhythm disorder is expected in the future by the increase in advancedaged and the progress of globalization of social structure, but it isthe present situation that a secure rhythm disorder improving agent isnot present. On the other hand, there are reports stating that brightlight therapy, including staring at a light of about 5,000 luxescontinuously for several hours in the early morning, shows excellenttherapeutic effect on nocturnal poriomania and delirium in patients withsenile dementia, rhythm disturbance in patients with delayed sleep phasesyndrome (DSPS) or the like (References 6, 7 and 9 to 12). However,since the bright light therapy which requires looking at a high luxlight source for a prolonged period of time is painful or burden forpatients and their caretakers, agents as substitutes for this lighttherapy are highly expected.

Based on tissue destruction and tissue transplantation experiments, ithas been found in 1972 that the rhythm center of mammals is present insuprachiasmatic nucleus (SCN) (References 13 and 14). However, molecularmechanism of the rhythm generation has been unclear until recent years(Reference 15). On the other hand, an arrhythmic mutant (Period mutant)of Drosophila melanogaster has been prepared by a genetic technique andthen a Period gene of Drosophila melanogaster has been cloned (Reference16, Reference 17). Since oscillatory expression of Period gene withabout 24 hour cycle is achieved in Drosophila melanogaster through themigration of translated Period protein (PERIOD) into the nucleus andsubsequent inhibition of its own transcription (negative feedbackmechanism), it is considered that outputs of circadian rhythm (behavior,timing of eclosion) are finally developed by this (References 18 and19). In mammals, on the other hand, human and mouse Period1 gene(Period1; Per1) has been cloned in 1997 as a homologue of the DrosophilaPeriod gene (Reference 20, Reference 21). Thereafter, mouse Period2 gene(Period2; Per2) (References 22 and 23) and mouse Period3 gene (Period3;Per3) (References 24 and 25) have been cloned. In addition to these,mouse Clock gene (Clock) (Reference 26) and mouse Bmal1 gene (Bmal1)(Reference 27) have been reported as mammalian clock genes. Thus, it ispossible for now to understand the rhythm generation mechanism at themolecular level. Actually, it has been found that the Clock gene andBmal1 gene are important for the circadian oscillation of a clock gene(References 44 and 45) and that a protein encoded by the Clock gene anda protein encoded by the Bmal1 gene bind to a CACGTG type E-box andactivate transcription of said clock gene, namely, the CACGTG type E-boxsequence is essential for the transcriptional activation by the CLOCKand BMAL1 (Reference 27).

A transgenic rat (mPer1; luc transgenic rat) harboring a DNA prepared byligating an upstream sequence of a mouse clock gene, Period1 gene (mPer1), with a luciferase gene has been reported in 2000 (Reference 28). Ithas been reported that the Period1 shows oscillatory expressions in notonly the suprachiasmatic nucleus but also in peripheral tissues of theliving body by measuring the luciferase activity in real time using aphotomultiplier tube detector (Photomal) (Reference 28). In addition,similar results have been reported also on a transgenic mouse (=Per1;luc transgenic mouse) harboring a DNA prepared by ligating an upstreamsequence of the Period1 gene with a luciferase gene (Reference 43).

It has been suggested that the Period2 gene among the three Period genehomologues takes an important role in the rhythm generation, because itscircadian rhythm disappears in mutant mice with artificial gene mutation(Reference 29). Also, it has been reported that the cause of a familialadvanced sleep phase syndrome (ASPS) is a point mutation of the Period2gene (Reference 30). Thus, the Period2 gene is a gene which not onlyshows abnormal rhythm in the mutant mice but also relates to rhythmdisorders in human has been confirmed.

Only the one report regarding the upstream region of Period2 gene is WO01/07654 on a mouse sequence, and said patent describes a DNA sequenceby defining it as a sequence which controls mouse Period2 transcriptionand describes about a method for identifying a Period2 transcriptioninhibitor, which comprises supplying a cell containing sequence (whichcontrols Per2 transcription)-linked reporter gene, introducing a testcompound and assaying transcription of the reporter gene. However, thereis no specific example on actually obtaining the above DNA sequencedescribed as a sequence which controls mouse Period2 transcription, andthere is no description such that it can be obtained. Also, there is nospecific example on the determination of transcription start site ormeasurement of transcriptional activity, too. In addition, there arepositions in the disclosed DNA sequence where bases cannot be specified,and sequence information regarding upstream sequence of the Period2 geneis not specifically disclosed.

Also, there are many reports on the analysis of the upstream sequence ofPeriod1 gene, but since Period1 and Period2 genes are located indifferent chromosomes and have no characteristic common sequence, it didnot become information for deducing the Period2 promoter sequence.

Great concern has been directed toward the development of a tool for thescreening of useful substances as rhythm disorder improving agentshaving a mechanism of function to control expression of biological clockgenes and a method for screening substances capable of controllingexpression of biological clock genes.

DISCLOSURE OF THE INVENTION

As a result of intensive studies, the present inventors have determinedfor the first time a human Period2 gene promoter sequence as a regionwhich controls transcriptional activity of the human Period2 gene andalso as a region that contributes to the oscillatory expression, therebyobtaining a construct containing the above promoter and a reporter geneand a cell containing the above construct. Also, a construct containingthe above promoter and a reporter gene and a cell containing the aboveconstruct were obtained by determining a mouse Period2 gene promotersequence. In addition, a transgenic animal harboring the above constructwas prepared. Next, in spite of the absence of the CACGTG type E-boxsequence in the mouse-derived Period2 promoter of the present invention,transcriptional activation of the Period2 gene by a heterodimer(BMAL1/CLOCK heterodimer) consisting of a protein encoded by a mouseBmal1 gene and a protein encoded by a mouse Clock gene and oscillatoryexpression were unexpectedly found. Also, a system by which promoteractivity of this gene can be easily detected was constructed. As aresult, the present invention provides a Period2 gene promoter, aconstruct containing the above promoter and a reporter gene, a cellcontaining the above construct and a transgenic animal harboring theabove construct, as tools useful for screening rhythm disorder improvingagents as substances which control expression and/or oscillatoryexpression of biological clock genes, and also provides a convenientmethod for screening rhythm disorder improving agents, and thus, thepresent invention has been completed. Also, the term “construct” as usedherein means a construct consisting of a DNA constructed by acombination of DNAs such that it shows the function of interest.

The CACGTG type E-box sequence is present in five positions of theupstream of the first exon of the mouse Period1 gene as one of thePeriod gene homologues, and the region which contributes to the basalactivity of Per1 transcription is present in a region containing firstexon, its environs and a human-mouse conserved segment of the firstintron, but it does not contain these five CACGTG type E-box sequences.The BMAL1/CLOCK heterodimer activates transcription of the mouse Period1gene by binding to the CACGTG type E-box sequence, but it hardly enhancethe transcriptional activity derived from the region which contributes abasal transcriptional activity and contains no CACGTG type E-boxsequence (Reference 27). Also, when the five CACGTG type E-boxes of themouse Period1 gene were mutated, the transcriptional activity in thepresence of the BMAL1/CLOCK heterodimer becomes a similar level of thebasal transcriptional activity (Reference 34). Based on these facts, itis considered that the BMAL1/CLOCK heterodimer induces transcriptionalactivation of the mouse Period1 gene via the CACGTG type E-box. On theother hand, it has been reported that the circadian oscillation ofPeriod1 and Period2 genes disappear under constant dark condition inBmal1 knockout mice (reference 44), and it has been reported that thecircadian oscillation of Period1 and Period2 genes attenuate underconstant dark condition in Clock/Clock mutant mice (reference 45). Thatis, it has been found that the Bmal1 and Clock are important for theoscillations. When considered from these results, the DNA sequencedefined as sequence, which controls mouse Period2 transcription, in theabove WO/01/07654 (that is, it corresponds to a part (from positions6,050 to 7,761) of a sequence consisting of nucleotides of positions4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1which is the DNA nucleotide sequence of the present invention, but is asequence having inconsistent sequences in several positions) merelyindicates a region having basal transcriptional activity because it doesnot contain the CACGTG type E-box. In the same manner, it was notconsidered that the DNA derived from Period2 gene of the presentinvention which does not contain the CACGTG type E-box will showtranscriptional enhancement by a BMAL1/CLOCK heterodimer, and itscontribution to the oscillatory expression could not be expected, too.However, it was unexpectedly found that the DNA of the present inventionwhich does not contain the CACGTG type E-box shows transcriptionalenhancement by a BMAL1/CLOCK heterodimer and also contributes to theoscillatory expression, namely that it is a region important for theoscillatory expression, so that a convenient system for measuring theoscillatory expression was constructed. Namely, since the Period2promoter of the present invention showed higher activity than that ofthe mouse Period2 gene sequence containing the CACGTG type E-box(namely, pCH1 of this specification), it was able to construct a moreeasily detectable system by using the DNA of the present invention andto provide a method for obtaining more useful rhythm improving agents.Thus, the present invention has been accomplished.

Accordingly, the present invention relates to

[1] A DNA which maintains a basal promoter activity and has a promoteractivity transcriptionally-activated by a BMAL1/CLOCK heterodimer, whichcomprises the nucleotide sequence described in the following (a), (b),(c), (d) or (e):

-   (a) a sequence consisting of nucleotides at positions 7,463 to 7,931    in the nucleotide sequence represented by SEQ ID NO:1,-   (b) a sequence consisting of nucleotides at positions 6,417 to 7,931    in the nucleotide sequence represented by SEQ ID NO:1,-   (c) a sequence consisting of nucleotides at positions 5,249 to 7,931    in the nucleotide sequence represented by SEQ ID NO:1,-   (d) a sequence consisting of nucleotides at positions 4,415 to 7,931    in the nucleotide sequence represented by SEQ ID NO:1, and-   (e) a sequence consisting of nucleotides at positions 3,820 to 6,068    in the nucleotide sequence represented by SEQ ID NO:2.

[2] A DNA which consists of the nucleotide sequence described in the(a), (b), (c), (d) or (e) according to [1].

[3] A construct which comprises the DNA according to [1] or [2] and areporter gene.

[4] A cell which comprises the construct according to [3].

[5] A method for screening a substance which controls expression ofPeriod2 gene, comprising the steps of:

-   -   allowing the cell according to [4] to contact with    -   a substance to be tested, and measuring a reporter activity.

[6] An transgenic animal transfected with the construct according to[3].

[7] The transgenic animal according to [6], wherein the animal is a rat.

[8] A method for screening a substance which controls expression and/oroscillatory expression of Period2 gene, comprising the steps of:

-   -   allowing the cell according to [4] or a suprachiasmatic nucleus        section or peripheral tissue of the transgenic animal according        to [6] or [7] to react with a substance to be tested, and    -   measuring oscillatory expression.

[9] A method for screening a substance which controls expression and/oroscillatory expression of Period2 gene, comprising the steps of:

-   -   administering a substance to be tested to the transgenic animal        according to [6] or [7], and    -   measuring oscillatory expression of suprachiasmatic nucleus of        the animal.

[10] The screening method according to any one of [5], [8] and [91,wherein the substance which controls expression and/or oscillatoryexpression of Period2 gene is a substance for improvement of rhythmdisorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of the sequence comparison between upstreamregions of mouse Period2 gene and human Period2 gene (hPer 2).

FIG. 2 shows nucleotide sequences of 7 segments highly coeserved betweenmouse and human.

FIG. 3 shows basal promoter activities of mouse Period2 gene upstreamregions.

FIG. 4 shows a function of a heterodimer of transcription factors,BMAL1/CLOCK, upon a vector pCH3 containing mouse Period2 gene upstreamregion.

FIG. 5 shows a result of automatic measurement of luminescence from asuprachiasmatic nucleus section, carried out for 10 days aftercommencement of the measurement.

FIG. 6 shows a part of the graph shown in FIG. 5 expanded only invertical direction.

FIG. 7 shows a result of automatic measurement of luminescence from aliver section, carried out for 10 days after commencement of themeasurement.

FIG. 8 shows a result of automatic measurement of luminescence from alung section, carried out for 10 days after commencement of themeasurement.

FIG. 9 shows a result of automatic measurement of luminescence from aneyeball, carried out for 10 days after commencement of the measurement.

FIG. 10 shows a result of automatic measurement of luminescence from apCH3-transfected culture cell, carried out for 4 days after commencementof the measurement.

FIG. 11 shows a result of automatic measurement of luminescence from apTM15-transfected culture cell, carried out for 5 days aftercommencement of the measurement.

FIG. 12 shows basal activities from various upstream regions of mousePeriod2 gene and functions of a heterodimer of transcription factors,BMAL1/CLOCK, upon vectors containing the same regions.

FIG. 13 shows a result of automatic measurement of luminescence from apCH3-D3-transfected culture cell, carried out for 6 days aftercommencement of the measurement.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained below in detail.

[DNA of the Present Invention]

The DNA of the present invention contains (a) a sequence consisting ofnucleotides at positions 7,463 to 7,931 in the nucleotide sequencerepresented by SEQ ID NO:1, (b) a sequence consisting of nucleotides atpositions 6,417 to 7,931 in the nucleotide sequence represented by SEQID NO:1, (c) a sequence consisting of nucleotides at positions 5,429 to7,931 in the nucleotide sequence represented by SEQ ID NO:1, (d) asequence consisting of nucleotides at positions 4,415 to 7,931 in thenucleotide sequence represented by SEQ ID NO:1, or (e) a sequenceconsisting of nucleotides at positions 3,820 to 6,068 in the nucleotidesequence represented by SEQ ID NO:2, and shows a Period2 gene promoteractivity.

The term “Period2 gene promoter activity” as used herein means apromoter activity which maintains at least a basal promoter activity asa DNA consisting of a sequence consisting of nucleotides at positions4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1(that is, having at least 50% of the activity of the basal promoteractivity of a DNA consisting of a sequence consisting of nucleotides atpositions 4,415 to 7,931 in the nucleotide sequence represented by SEQID NO:1, preferably having substantially the same level of basalpromoter activity of the DNA or superior to that) and is also enhancedby BMAL1/CLOCK heterodimer on transcriptional activity (namely tocontribute to oscillatory expression).

The term “basal promoter activity” as used herein means a promoteractivity when a predetermined period of time (e.g., 48 hours) is passedunder no stimulus condition, and the term “no stimulus condition” meansspecifically conditions in the absence of a BMAL1/CLOCK heterodimer asshown in Example 3. Also, the above “BMAL1/CLOCK heterodimer” istranscription factors which controls each transcription of the Period1gene, Period2 gene and Period3 gene.

Although the method for judging whether or not a certain DNA shows“Period2 gene promoter activity” is not particularly limited, it can bejudged, for example, by verifying that it is substantially the same asor superior to the promoter activity of the DNA consisting of a sequenceconsisting of nucleotides at positions 4,415 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1, through the measurement of basalpromoter activity in the absence of the BMAL1/CLOCK heterodimer as shownin Example 3, and further verifying whether or not it showsdose-dependent transcriptional activation by a BMAL1/CLOCK heterodimer.

The desirable DNA of the present invention includes a DNA consisting ofa sequence consisting of nucleotides at positions 7,463 to 7,931 in thenucleotide sequence represented by SEQ ID NO:1, a sequence consisting ofnucleotides at positions 6,417 to 7,931 in the nucleotide sequencerepresented by SEQ ID NO:1, a sequence consisting of nucleotides atpositions 5,429 to 7,931 in the nucleotide sequence represented by SEQID NO:1 or a sequence consisting of nucleotides at positions 4,415 to7,931 in the nucleotide sequence represented by SEQ ID NO:1, or a DNAconsisting of a sequence consisting of nucleotides at positions 3,820 to6,068 in the nucleotide sequence represented by SEQ ID NO:2. However,any DNA is included in the DNA of the present invention, so long as theDNA contains a DNA consisting of a sequence consisting of nucleotides atpositions 7,463 to 7,931 in the nucleotide sequence represented by SEQID NO:1, a sequence consisting of nucleotides at positions 6,417 to7,931 in the nucleotide sequence represented by SEQ ID NO:1, a sequenceconsisting of nucleotides at positions 5,429 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1 or a sequence consisting ofnucleotides at positions 4,415 to 7,931 in the nucleotide sequencerepresented by SEQ ID NO:1, or a sequence consisting of nucleotides atpositions 3,820 to 6,068 in the nucleotide sequence represented by SEQID NO:2, and has a Period2 gene promoter activity.

As is shown later in Example 1, the sequence consisting of nucleotidesat positions 4,415 to 7,931 in the nucleotide sequence represented bySEQ ID NO:1 is an upstream region of the mouse Period2 gene and, as isshown later in Example 3, has a Period2 gene promoter activity.

As is shown later in Example 1, the DNA consisting of a sequenceconsisting of nucleotides at positions 3,820 to 6,068 in the nucleotidesequence represented by SEQ ID NO:2 is an upstream region of the humanPeriod2 gene. From the experimental results on mouse genes (Examples 1and 3 which are described later), it was found that the mouse DNAshowing promoter activity (namely the DNA consisting of a sequenceconsisting of nucleotides at positions 4,415 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1) is a DNA resulting from theelimination of the region containing conserved segments (IV, V, VI andVII) in the first intron of Period2 gene from the DNA containing thehuman/mouse seven conserved segments. Accordingly, it is considered thata human DNA corresponding to the DNA resulting from the elimination ofthe region containing conserved segments (IV, V, VI and VII) in thefirst intron of Period2 gene from the DNA containing the human/mouseseven preserved regions, namely the DNA consisting of a sequenceconsisting of nucleotides at positions 3,820 to 6,068 in the nucleotidesequence represented by SEQ ID NO:2, also has a Period2 gene promoteractivity.

Although not particularly limited, the DNA of the present invention canbe prepared, for example, by (1) using polymerase chain reaction (PCR)method or by (2) screening a phage library.

(1) Polymerase Chain Reaction (PCR) Method

When the DNA of the present invention is prepared using the PCR method,a primer set capably of amplifying the DNA of the present invention isfirstly designed based on the information on each nucleotide sequencerepresented by SEQ ID NO:1 or 2.

In the case of a DNA of the present invention containing a sequenceconsisting of nucleotides at positions 4,415 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1, which is the mouse Period2 genepromoter, a primer set is designed based on the information on thenucleotide sequence represented by SEQ ID NO:1 in such a manner that theamplified product contains the sequence consisting of nucleotides atpositions 4,415 to 7,931 in the nucleotide sequence represented by SEQID NO:1 but does not contain in and after the conserved segment IV(position 8,534). Also, in the case of a DNA of the present inventioncontaining a sequence consisting of nucleotides at position 3,820 to6,068 in the nucleotide sequence represented by SEQ ID NO:2, which isthe human Period2 gene promoter, a primer set is designed based on theinformation on the nucleotide sequence represented by SEQ ID NO:2 insuch a manner that the amplified product contains a sequence consistingof nucleotides at positions 3,820 to 6,068 in the nucleotide sequencerepresented by SEQ ID NO:2 but does not contain in and after theconserved segment IV (position 6,531).

The DNA of the present invention can be obtained by carrying out PCRusing the thus designed respective primer set and a genomic DNA as thetemplate.

(2) Phage Library Screening Method

When the DNA of the present invention is prepared by screening a phagelibrary (e.g., Maniatis, T. et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, NY, 1982), a probe which canscreen phage clones containing the DNA of the present invention isfirstly designed base on the information on each nucleotide sequence ofSEQ ID NO:1 or 2.

In the case of a DNA of the present invention containing a sequenceconsisting of nucleotides at positions 4,415 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1, which is the mouse Period2 genepromoter, a probe is designed based on the information on the sequenceconsisting of nucleotides at positions 4,415 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1. Also, in the case of a DNA of thepresent invention containing a sequence consisting of nucleotides atpositions 3,820 to 6,068 in the nucleotide sequence represented by SEQID NO:2, which is the human Period2 gene promoter, a probe is designedbased on the information on the sequence consisting of nucleotides atpositions 3,820 to 6,068 in the nucleotide sequence represented by SEQID NO:2.

A phage clone containing the DNA of the present invention can beobtained by screening a phage library using the thus designed respectiveprobe. The DNA of the present invention can be obtained by treating thethus obtained phage clone with appropriate restriction enzymes and thenpurifying a DNA fragment of interest using an appropriate purificationmeans (e.g., agarose gel electrophoresis).

[Construct and Cell of the Present Invention]

The construct of the present invention contains the DNA of the presentinvention and a reporter gene. As the reporter gene, a gene encoding aknown reporter protein which can be used as an index of gene expressionin the cells can be used. The reporter protein includes luciferase,secretion type alkaline phosphatase (SEAP), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS),β-D-galactosidase, aequorin and the like. According to the construct ofthe present invention, it is preferable to use a luciferase gene as thereporter gene.

According to the construct of the present invention, arranging positionsof the DNA of the present invention and a reporter gene are not limited,so long as the reporter gene is arranged in a downstream position of theDNA of the present invention and under control of the promoter activityof the DNA of the present invention. Also, the construct of the presentinvention is not particularly limited, so long as the construct containsat least the DNA of the present invention and a reporter gene, but it ispreferable that it further contains a vector region.

Although the construct of the present invention is not particularlylimited, a construct of the present invention further containing avector region in addition to the DNA of the present invention and areporter gene can be prepared, for example, by introducing the DNA ofthe present invention into a multi-cloning site of an appropriatereporter vector (namely, a vector containing a reporter gene). Thereporter vector includes a vector pGL3-basic containing a gene encodingluciferase (Promega), a vector pSEAP2-basic containing a gene encodingSEAP (Clontech), and a vector pd1EGFP containing a gene encoding labiletype GFP (Clontech).

More specifically, the construct of the present invention can beprepared by introducing the DNA of the present invention obtained byusing the PCR method into a multi-cloning site of a reporter vector.

Also, the construct of the present invention can be prepared byintroducing the DNA of the present invention, which has been prepared bytreating a phage clone obtained by screening a phage library withappropriate restriction enzymes and then purifying it using anappropriate purification means (e.g., agarose gel electrophoresis), ifnecessary further subjecting to a blunt-ended treatment, into amulti-cloning site of a reporter vector. For example, in the case of aphage clone containing a sequence consisting of nucleotides at positions3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2,which is the human Period2 gene promoter, the construct of the presentinvention containing the human Period2 gene promoter can be prepared bytreating the phage clone with appropriate restriction enzymes (e.g., acombination of a restriction enzyme Aor51HI which cuts at position 3,513position and a restriction enzyme PshBI which cuts at position 6,447),obtaining a 2,935 bp DNA fragment by purifying it using an appropriatepurification means (e.g., an agarose gel electrophoresis), smooth-endingthe DNA fragment and then introducing it into a multi-cloning site of areporter vector. A deletion construct containing the DNA of the presentinvention can be prepared, for example, by treating a longer DNA amongthe DNAs of the present invention obtained by the above method withappropriate restriction enzymes and purifying the digest by anappropriate purification method and then subjecting the thus obtainedDNA fragment of interest to self-ligation. More specifically, it can beobtained by the method described in Example 10.

The cell of the present invention contains the construct of the presentinvention. Although not particularly limited, the cell of the presentinvention can be prepared by transforming an appropriate host cell(preferably a eucaryote) with the construct of the present invention(preferably, a construct of the present invention further containing avector region in addition to the DNA of the present invention and areporter gene).

The host cell of eucaryote includes cells such as vertebrate, insect andyeast, and examples of the vertebrate cells include mouse NIH3T3 cell,monkey COS cell (Reference 37), Chinese hamster ovary cell (CHO)dihydrofolate reductase deficient strain (Reference 38), mouse L cell,mouse A9 cell, monkey BS-C-1 cell and the like, but it is preferable touse mouse NIH3T3 cell.

The construct of the present invention can be incorporated into a hostcell by, for example, a DEAE-dextran method (Reference 39), a calciumphosphate-DNA coprecipitation method (Reference 40), a method usingcommercially available transfection reagents [e.g., Lipofectamine 2000(GIBCO-BRL), FuGENE™6 Transfection Reagent (Roche Diagnostics)],electroporation (Reference 41) or the like.

The cell of the present invention can be cultured in accordance with aconventional method. As the medium which can be used in the culturing,various generally used media can be appropriately selected in responseto the host cell employed. For example, in the case of the NIH3T3 cell,a medium prepared by supplementing DMEM (Dulbecco's modified Eagle'smedium) with glucose (final concentration=4.5 g/l) and fetal bovineserum (final concentration=10%) can be used.

[Transgenic Animal of the Present Invention]

The transgenic animal of the present invention is not particularlylimited, so long as the animal is transfected with the construct of thepresent invention, but it can be prepared based on a conventionallyknown method (e.g., Reference 35), except that the construct of thepresent invention is used as the DNA to be transfected. Specifically, itcan be prepared based on the procedure described later in Example 4.

Also, the term “animal” as used herein means an animal excluding human(namely non-human animal), and examples include mammals excluding human(e.g., rat, mouse, dog, cat, monkey, pig, cattle, sheep, rabbit, goat,dolphin or horse), birds (e.g., domestic fowl or quail), amphibia (e.g.,frog), reptiles, insects and the like, and rat and mouse are preferred,and rat is particularly preferred.

[Screening Method of the Present Invention]

The screening method of the present invention can be carried out usingthe cell of the present invention, a suprachiasmatic nucleus section orperipheral tissue of the transgenic animal of the present invention orthe transgenic animal of the present invention itself. Although the testsubstance to be used in the screening is not particularly limited,examples include commercially available compounds (including peptides),various known compounds (including peptides) registered in chemicalfile, compounds obtained by combinatorial chemistry techniques(Reference 31), culture supernatants of microorganisms, naturalcomponents derived from plants and marine organisms, animal tissueextracts or compounds (including peptides) prepared by chemically orbiologically modifying the compounds (including peptides) selected bythe screening method of the present invention.

According to the screening method of the present invention which usesthe cell of the present invention, a substance which controls expressionof the Period2 gene, namely a substance which modifies its transcriptionactivity, can be selected by allowing the cell of the present inventionto contact with a substance to be tested and measuring the reporteractivity (namely, a reporter assay).

When the cell of the present invention is allowed to contact with asubstance to be tested, a cell into which the construct is temporarilysort stably introduced is prepared and drug (namely substance to betested) stimulation is carried out. A substance which can controlexpression of the Period2 gene can be screened by carrying out areporter assay after a predetermined period of time of the drug (namelysubstance to be tested) stimulation.

The reporter assay can be carried out by a known assay method inresponse to the kinds of a reporter protein to be used. For example,when a firefly luciferase is used as the reporter protein, luciferin canbe used as its chemical substrate for luciferin-luciferase luminescence,and when a Renilla luciferase derived from sea pansy is used,coelenteradin can be used as its chemical substrate forluciferin-luciferase luminescence. Also, when SEAP is used, CSPD[disodium3-(methoxyspiro(1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13.7]decan)-4-yl)phenylphosphate] and MUP (4-methylunbellifery phosphate) can be used as itschemical substrates for the respective luminescent and fluorescentassay. A luciferase assay is preferred as the reporter assay, and theluciferase assay can be carried out preferably under the conditionsdescribed in Example 3 which is described later.

All of the substances which enhance expression of the Period2 gene andthe substances which inhibit the same are useful as agents improvingrhythm disorders, and when a substance which inhibits expression of thePeriod2 gene is screened, the screening can be effected by an assay inthe coexistence of a known Period2 gene transcription activating factorsuch as BMAL1/CLOCK heterodimer. A synchronizing factor during daytimehas a property to accelerate transcription or release the transcriptioninhibition (transcription acceleration as a consequence) of the Period2gene, and it can synchronized the circadian rhythm when reacted indaytime and causes phase shift when reacted in night. Also, asynchronizing factor during night has a property to inhibittranscription or release the transcription acceleration (transcriptioninhibition as a consequence) of the Period gene, and it has beenconfirmed that it can synchronized the rhythm when reacted in night andcauses phase shift when reacted in daytime. Accordingly, an agent whichaccelerates transcription of the Period2 gene can synchronize the rhythmwhen taken during the day, and on the contrary, an agent which inhibitstranscription of the Period2 gene can synchronize the rhythm when takenduring the night.

According to the screening method of the present invention which usesthe cell of the present invention or a suprachiasmatic nucleus sectionor peripheral tissue of a transgenic animal, a substance which controlsexpression of the Period2 gene can be selected by allowing the cell ofthe present invention or a suprachiasmatic nucleus section or peripheraltissue of a transgenic animal to react with a substance to be tested andmeasuring the oscillatory expression.

There is a report stating that oscillatory curves with circadian cyclecan be obtained by automatic measurement of luminescence level from asuprachiasmatic nucleus section or peripheral tissue, under culturing,of a mPer1:luc transgenic rat using a photomultiplier tube detector(Photomal) (Reference 28). When the oscillation of luminescence from asuprachiasmatic nucleus section or peripheral tissue of a transgenicanimal harboring a construct containing a DNA showing Period2 genepromoter activity and a reporter gene is automatically measured usingthis method, and the oscillation of the luminescence becomes stable, asubstance to be tested (e.g., a substance to be tested such as acandidate for an improving agent of biological rhythm screened by thescreening method of the present invention which uses the cell of thepresent invention) is allowed to act upon the above suprachiasmaticnucleus section or peripheral tissue of a transgenic animal on one hand,and, as a control, a solvent [e.g., dimethyl sulfoxide (DMSO) or thelike] alone of the substance to be tested is allowed to act upon thesuprachiasmatic nucleus section or peripheral tissue on the other hand,and the automatic measurement is continued. As the transgenic animal, atransgenic rat is desirable. Phase shift (positional change inoscillation peak or bottom), namely time delay or time advance ofoscillatory expression, can be evaluated by comparing the oscillatorycurve of luminescence obtained from the group treated with a substanceto be tested (e.g., a biological rhythm disorder-improving candidatesubstance to be tested) with the oscillatory curve of luminescenceobtained from the untreated control group. A substance which controlsexpression and/or oscillatory expression of the Period2 gene can beselected by screening substances showing ideal phase shift in thismanner. In addition, the above screening can also be carried out usingthe cell of the present invention, that is, a cell into which a DNAprepared by ligating an upstream sequence of the mouse Period2 gene witha reporter gene is introduced, instead of a suprachiasmatic nucleussection or peripheral tissue, and synchronizing the cells The cells canbe synchronized by stimulation with high concentration serum ordexamethasone (DEX), but preferably can be synchronized by the methoddescribed in Example 6.

For example, a substance which controls expression and/or oscillatoryexpression of the Period2 gene can be selected by carrying out theautomatic measurement of luminescence level from a suprachiasmaticnucleus section or peripheral tissue (e.g., liver, kidney, lung oreyeball) of a transgenic rat (mPer2:luc transgenic rat) prepared byintroducing into a rat a DNA in which an upstream sequence of the mousePeriod2 gene is ligated with a luciferase gene, by the method describedin Example 5 which is described later, and evaluating the phase shift bycomparing the oscillation curves of luminescence level obtained from thetest substance-treated group with those obtained from the untreatedgroup (control). When it is carried out by the method described inExample 5, it is possible to evaluate the function upon the rhythm ofsubstances to be tested, by changing one suprachiasmatic nucleus sectionto a fresh medium to which a solvent (e.g., DMSO, etc.) alone is addedas a control, and the other to a fresh medium to which a substance to betested dissolved in a solvent (e.g., DMSO, etc.) is added, at a stage offrom the 3rd to 9th day after commencement of the measurement duringwhich the oscillatory expression is observed, and comparing theoscillation after addition of the substance to be tested with that ofthe control. Also, regarding peripheral tissues, it is possible to carryout screening of substances to be tested by changing medium of, forexample liver, lung and eyeball, to a medium containing a substance tobe tested (a solvent alone in the case of control) in the same manner,during the 4th and 5th days after commencement of the measurement,during the 1st to 5th days after commencement of the measurement andduring the 1st to 3rd days after commencement of the measurement,respectively, during which the oscillatory expression is observed.

Also, as shown in the following Example 5, in peripheral tissues,oscillation rhythm occurred several times and then attenuated byintercellular de-synchronization (cf. FIGS. 7 to 9), and it is possibleto obtain an agent showing a function to improve attenuated locomoteractivity rhythm and/or sleep rhythm in the aged by screening an agentwhich inhibits this attenuation.

Also, when a large peripheral tissue (e.g., liver, lung, etc.) is usedin the screening, it is desirable to a peripheral tissue section.

According to the screening method of the present invention which usesthe transgenic animal of the present invention, a substance whichcontrols expression and/or oscillatory expression of the Period2 genecan be selected by administering a substance to be tested to thetransgenic animal of the present invention and measuring oscillatoryexpression in the suprachiasmatic nucleus of the animal.

Specifically, a substance to be tested (e.g., a substance to be testedsuch as a biological rhythm improving agent candidate screened by thescreening method of the present invention which uses the cell of thepresent invention) is administered to a transgenic animal harboring aconstruct containing a DNA showing the Period2 promoter activity and areporter gene. When luciferase is used as the reporter protein, it ispossible to screen a compound by evaluating its function uponoscillation of the rhythm center suprachiasmatic nucleus, by the use ofa method in which luciferin is supplied from the vertex into thevicinity of the suprachiasmatic nucleus through a capillary and theluminescence level is simultaneously and continuously measured through amicro-optical fiber inserted into just above the suprachiasmatic nucleuswhile keeping the individual alive (Reference 42).

EXAMPLES

The present invention is specifically described based on examples, butthey do not limit the scope of the present invention. Also, unlessotherwise indicated, these were carried out in accordance with knownmethods (e.g., Maniatis, T. et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, NY., 1982; and Hille, B., IonicChannels of Excitable Membranes, 2nd Ed., Sinauer Associates Inc., MA,1992).

Example 1

<Preparation of Mouse and Human Period2 Upstream DNA Sequences andComparative Analysis>

Preparation of a mouse Period2 upstream sequence was carried out by ascreening using a mouse genomic DNA phage library and using GenomeWalker Kit (Clontech).

Firstly, polymerase chain reaction (PCR) was carried out by preparing aprimer set which can amplify a fragment between a position 218 and aposition 723 in mouse Period2 gene cDNA from the reported sequence ofmouse Period2 gene (GenBank AFO36893), namely a forward primerconsisting of the nucleotide sequence represented by SEQ ID NO:3 [asequence at positions 218 to 239 in the mouse Period2 gene cDNA (GenBankAFO36893)] and a reverse primer consisting of the nucleotide sequencerepresented by SEQ ID NO:4 [a sequence complimentary to the sequence atpositions 702 to 723 in the mouse Period2 gene cDNA (GenBank AFO36893)].In this case, the above PCR was carried out using a Taq polymerase(AmpliTaq Gold; Applied Biosystems) as the enzyme and repeating 40 timesof a cycle consisting of incubation at 95° C. (10 minutes) andsubsequent incubation at 94° C. (15 seconds), 60° C. (30 seconds) and72° C. (1 minute).

Using the thus obtained amplified fragment (505 bp) as the probe,screening from a mouse genomic DNA phage library was carried out. As aresult, a clone containing the first intron of the mouse Period2 gene6.0 kb upstream from the second exon was obtained. When a sequencingreaction was carried out on the thus obtained phage DNA using a DNAsequencing reagent (BigDye Terminator Cycle Sequencing FS Ready ReactionKit; Applied Biosystems) in accordance with its manufacture'sinstructions and then the DNA nucleotide sequence was analyzed using aDNA sequencer (ABI PRISM 377; Applied Biosystems), a sequence consistingof nucleotides at positions 10,104 to 17,004 in the nucleotide sequencerepresented by SEQ ID NO:1 was obtained.

Next, in order to obtain a further upstream sequence from the phage DNAsequence, preparation of the upstream sequence was carried out usingGenome Walker Kit (Clontech). Libraries MDL1 to MDL5 attached to the kitwere used as the template of the Genome Walker Kit, and a Taq DNApolymerase (Advantage Genomic polymerase Mix; Clontech) was used as theenzyme for PCR.

Firstly, using AP1 attached to the kit and a first primer consisting ofthe nucleotide sequence represented by SEQ ID NO:5 (a sequencecomplimentary to a sequence consisting of nucleotides at positions13,005 to 13,034 in the nucleotide sequence represented by SEQ ID NO:1)as a primer set, PCR was carried out in the presence of 5% dimethylsulfoxide (DMSO) by repeating 7 times of a cycle consisting of 94° C. (2seconds) and 72° C. (3 minutes) and subsequent 36 times of a cycleconsisting of 94° C. (2 seconds) and 67° C. (3 minutes) and then finallyincubating at 67° C. for 4 minutes. Next, using 1 μl of 50 times dilutedsolution of the thus obtained reaction product as the template and usingAP2 attached to the kit and a second primer consisting of the nucleotidesequence represented by SEQ ID NO:6 (a sequence complimentary to asequence consisting of nucleotides at positions 12,429 to 12,458 in thenucleotide sequence represented by SEQ ID NO:1) as a primer set, PCR wascarried out by repeating 5 times of a cycle consisting of 94° C. (2seconds) and 72° C. (3 minutes) and subsequent 24 times of a cycleconsisting of 94° C. (2 seconds) and 67° C. (3 minutes) and then finallyincubating at 67° C. for 4 minutes.

A sequence consisting of nucleotides at positions 9,870 to 12,458 in thenucleotide sequence represented by SEQ ID NO:1 was obtained bysequencing analysis of the amplified band obtained by the above two stepPCR. Based on this sequence, a primer set for obtaining a furtherupstream sequence, namely a first primer consisting of the nucleotidesequence represented by SEQ ID NO:7 (a sequence complimentary to asequence consisting of nucleotides at positions 10,103 to 10,132 in thenucleotide sequence represented by SEQ ID NO:1) and a second primerconsisting of the nucleotide sequence represented by SEQ ID NO:8 (asequence complimentary to a sequence consisting of nucleotides atpositions 10,021 to 10,050 in the nucleotide sequence represented by SEQID NO:1), were prepared.

The above two step PCR was repeated except that a first primerconsisting of the nucleotide sequence represented by SEQ ID NO:7 and asecond primer consisting of the nucleotide sequence represented by SEQID NO:8 were used instead of the first primer consisting of thenucleotide sequence represented by SEQ ID NO:5 and the second primerconsisting of the nucleotide sequence represented by SEQ ID NO:6, andsequencing analysis of the thus obtained amplified band was carried outto obtain a sequence consisting of nucleotides at positions 9,146 to10,050 in the nucleotide sequence represented by SEQ ID NO:1.

Subsequently, in order to obtain further upstream sequences one by one,the above procedures, namely preparation of first primer and secondprimer, two step PCR and sequencing analysis of obtained amplified band,were repeated. Combination of the first primer and second primer used ineach two step PCR was, a combination of a first primer consisting of thenucleotide sequence represented by SEQ ID NO:9 (a sequence complimentaryto a sequence consisting of nucleotides at positions 9,355 to 9,384 inthe nucleotide sequence represented by SEQ ID NO:1), and

-   -   a second primer consisting of the nucleotide sequence        represented by SEQ ID NO:10 (a sequence complimentary to a        sequence consisting of nucleotides at positions 9,307 to 9,336        in the nucleotide sequence represented by SEQ ID NO:1);    -   a combination of a first primer consisting of the nucleotide        sequence represented by SEQ ID NO:11 (a sequence complimentary        to a sequence consisting of nucleotides at positions 7,838 to        7,857 in the nucleotide sequence represented by SEQ ID NO:1),        and a second primer consisting of the nucleotide sequence        represented by SEQ ID NO:12 (a sequence complimentary to a        sequence consisting of nucleotide sequences at positions 7,818        to 7,837 in the nucleotide sequence represented by SEQ ID NO:1);        and    -   a combination of a first primer consisting of the nucleotide        sequence represented by SEQ ID NO:13 (a sequence complimentary        to a sequence consisting of nucleotides at positions 2,234 to        2,263 in the nucleotide sequence represented by SEQ ID NO:1),        and a second primer consisting of the nucleotide sequence        represented by SEQ ID NO:14 (a sequence complimentary to a        sequence consisting of at positions 2,134 to 2,163 in the        nucleotide sequence represented by SEQ ID NO:1), and used in        this order.

Also, a further upstream sequence was not able to be obtain by the twostep PCR using primers designed based on a sequence consisting ofnucleotides at positions 8,918 to 9,336 in the nucleotide sequencerepresented by SEQ ID NO:1, which was obtained as a result of thesequencing analysis of a DNA band amplified by the two step PCR using acombination of the first primer consisting of the nucleotide sequencerepresented by SEQ ID NO:9 and the second primer consisting of thenucleotide sequence represented by SEQ ID NO:10. Accordingly, in orderto obtain a further upstream sequence, the two step PCR was carried outusing a combination of a first primer consisting of the nucleotidesequence represented by SEQ ID NO:11 and a second primer consisting ofthe nucleotide sequence represented by SEQ ID NO:12, which had beendesigned based on the first exon sequence.

As a result of the analysis so far carried out, a sequence consisting ofnucleotides at positions 1 to 17,004 in the nucleotide sequencerepresented by SEQ ID NO:1 (however, excluding a sequence consisting ofnucleotides at positions 7,858 to 9,145) was obtained. In order tocompensate the gap between the first exon and the first intron, whichcorresponds to the undetermined sequence consisting of nucleotides atpositions 7,858 to 9,145, two step PCR (nested PCR) was carried outusing a mouse genomic DNA as the template and using a Taq DNA polymerase(Advantage Genomic polymerase Mix; Clontech) as the enzyme. Using aforward primer consisting of the nucleotide sequence represented by SEQID NO:15 (a sequence consisting of nucleotides at positions 7,516 to7,545 in the nucleotide sequence represented by SEQ ID NO:1) and areverse primer consisting of the nucleotide sequence represented by SEQID NO:16 (a sequence complimentary to a sequence consisting ofnucleotides at positions 9,063 to the 9,092 in the nucleotide sequencerepresented by SEQ ID NO:1) as a primer set, the first PCR was carriedout in the presence of 5% DMSO by incubating at 95° C. for 1 minute,repeating 35 times of a cycle consisting of 94° C. (15 seconds), 60° C.(30 seconds) and 68° C. (5 minutes) and then finally incubating at 68°C. for 10 minutes. The second PCR was carried out under the sameconditions as in the first PCR, except that a forward primer consistingof the nucleotide sequence represented by SEQ ID NO:17 (a sequenceconsisting of at positions 7,556 to 7,585 in the nucleotide sequencerepresented by SEQ ID NO:1) and a reverse primer consisting of thenucleotide sequence represented by SEQ ID NO:18 (a sequencecomplimentary to a sequence consisting of positions 8,977 to 9,006 inthe nucleotide sequence represented by SEQ ID NO:1) were used as aprimer set.

A 1.4 kbp DNA fragment was obtained as a result of the above two stepPCR, and by its sequence analysis, it was able to compensate the gapbetween the first exon and the first intron.

The nucleotide sequence represented by SEQ ID NO:1 was obtained bysummarizing all of the above analyzed results. It was found that thefirst exon (positions 7,736 to 7,857), the second exon (positions 16,158to 16,396) and the first intron (positions 7,858 to 16,157) are includedin the nucleotide sequence represented by SEQ ID NO:1, and that thetranslation start site of the mouse Period2 is present in the secondexon (position 16,176). Also, the transcription start site is present atposition 7,736 (cf. the following Example 2).

Since it is expected that a region important for the regulation of geneexpression is conserved between species when upstream sequences of mouseand human genes are compared, an attempt was made to obtain an upstreamsequence of the human Period2 gene (GenBank NM-00389). As a result ofthe retrieval of a draft sequence from a net [BLAST 2; National Centerfor Biotechnology Information (NCBI)], a clone containing an upstreamregion of the human Period2 gene (GenBank AC013400.5; 188.2 kbp;phase 1) was found.

Since there was a gap between the first intron and the second exon inthis draft sequence clone AC013400.5, PCR was carried out using a humangenomic DNA (Clontech) as the template. Using a forward primerconsisting of the nucleotide sequence represented by SEQ ID NO:19 (asequence consisting of nucleotides at positions 14,577 to 14,606 in thenucleotide sequence represented by SEQ ID NO:2) and a reverse primerconsisting of the nucleotide sequence represented by SEQ ID NO:20 (asequence complimentary to a sequence consisting of nucleotides atpositions 15,855 to 15,884 in the nucleotide sequence represented by SEQID NO:2) as a primer set and using a Taq DNA polymerase (AmpliTaq Gold;Applied Biosystems) as the enzyme, the above PCR was carried out byincubating at 95° C. for 10 minutes and then repeating 40 times of acycle consisting of 94° C. (15 seconds), 60° C. (30 seconds) and 72° C.(2 minutes), and the DNA sequence of a DNA fragment containing the gap(about 1.2 kbp) was analyzed.

On the other hand, since it was found thereafter that a new clone(GenBank AC012485.13; 168.7 kbp; phase 3 complete sequence) wasregistered on the net, a region of 17,112 bp (a sequence complimentaryto positions 35,354 to 52,465 in the GenBank AC012485.13) correspondingto the 17,004 bp of the above mouse genomic sequence was extracted fromthe clone GenBank AC012485.13 and shown as SEQ ID NO:2. From theanalysis of the thus obtained upstream sequence of the human Period2gene, it was found that the human translation start site is also presentin the second exon similar to the case of mouse. Thus, sequenceinformation on the about 17 kbp upstream from the second exon extendingabout 17 kbp of the mouse Period2 gene and human Period2 gene wasobtained.

The thus obtained sequence information on the upstream regions extendingabout 17 kbp of the mouse Period2 gene and human Period2 gene wassubjected to homology comparison analysis (analyzing conditions aredefault values) using BLAST 2 SEQUENCES(http://www.nobi.nlm.nih.gov.blast/b12seg/b12.html) VERSION BLASTN2.2.1AUG. 1, 2001 program of NCBI. A region having a high Expect accuracy ofe-3 or less was defined as positive in preservation ability. Results ofthe analysis are shown in FIG. 1. As shown in FIG. 1, it was found thatseven fragmentarily conserved segments (I to VII) are present in beforeand after the first exon between human and mouse.

Respective sequences in the conserved segments are shown in FIG. 2. InFIG. 2, the symbol “H” indicates human sequence, the symbol “M”indicates mouse sequence and the “|” between human sequence and mousesequence indicates a position where the kind of base coincided betweenhuman and mouse.

By summarizing all results of the above analysis, it was found that thefirst intron (positions 6,069 to 16,461) and the second exon (positions16,462 to 16,710) are contained in the nucleotide sequence representedby SEQ ID NO:2, and the human Period2 translation start site is presentat position 16,481.

When an upstream region DNA fragment is obtained by PCR using a genomicDNA as the template, there is a possibility of causing a gene mutation.An attempt was made to clone a phage containing an upstream region DNAfragment having no mutation from 1×10⁶ phage particles in accordance themanufacture's instructions of a mouse genomic phage library (Clontech).

In order to prepare a probe, a primer consisting of the nucleotidesequence represented by SEQ ID NO:17 and a primer consisting of thenucleotide sequence represented by SEQ ID NO:18 were designed based onthe mouse Period2 gene upstream sequence information obtained in theabove. Using a mouse genomic DNA (Clontech) as the template and using aTaq DNA polymerase (Advantage Genomic DNA polymerase Mix; Clontech) asthe enzyme, PCR was carried out by incubating at 95° C. for 1 minute andthen repeating 35 times of a cycle consisting of 95° C. (15 seconds),60° C. (30 seconds) and 68° C. (2 minutes), thereby obtaining a DNAfragment of 1.4 kb.

The thus obtained DNA fragment was cloned into the EcoRV site of aplasmid (pBluescript; STRATAGENE) and amplified. This plasmid wassubjected to restriction enzyme NotI/SalI treatment and then to anagarose gel electrophoresis to obtain a DNA fragment for probe. Next,the DNA fragment for probe was labeled using [α-³²P]dCTP (AmershamPharmacia Biotech) in accordance with the manufacture's instructions ofa labeling kit (BcaBEST Labeling Kit; Takara Shuzo). As a result of thescreening of the mouse genomic phage library using this probe, a clonecontaining six conserved segments between human and mouse was obtained.This phage clone in a large amount was prepared, and the phage DNAcontaining the upstream region was extracted using Phage DNA ExtractionKit (QIAGEN) in accordance with the manufacture's instructions.

In order to obtain a DNA fragment containing seven conserved regions,the thus obtained phage genome was digested with restriction enzymesEcoT221 and NheI and then subjected to an agarose gel electrophoresis toextract and purify a band of a 6.4 kb DNA fragment. The nucleotidesequence of the above DNA fragment is a sequence consisting ofnucleotides at positions 4,415 to 10,877 in the nucleotide sequencerepresented by SEQ ID NO:1. Blunt-ending of the purified DNA fragmentwas carried out using DNA Blunting Kit (Takara Shuzo).

Since a luciferase vector pGL3-basic (Promega; to be referred to aspGL3-b hereinafter) is a reporter vector having no promoter sequence, itcan evaluate the promoter activity of upstream fragments. A vector pCH1was prepared by digesting this vector pGL3-b with a restriction enzymeSmaI and then inserting the mouse upstream fragment which had beenblunt-ended in advance. Subsequently, a vector pCH3 in which theconserved segments (IV, V, VI, VII) in the first intron were removedfrom the vector pCH1 was prepared by subjecting the vector pCH1 to atreatment with restriction enzymes, XhoI and SnaBI, removing the DNAfragment including the conserved segments (IV, V, VI, VII) by an agarosegel electrophoresis and then carrying out self-ligation. The upstreamregion sequence of the mouse Period2 gene in the vector pCH3 correspondsto a sequence consisting of nucleotides at positions 4,415 to 7,931 inthe nucleotide sequence represented by SEQ ID NO:1.

Example 2

<Identification of Transcription Start Site of Mouse Period2>

Whether or not the vector pCH1 and vector pCH3 obtained in Example 1containing the mouse Period2 gene upstream region fragment contain apromoter region can be verified by examining the presence of thetranscription start site. In order to identify the transcription startsite, an attempt was made to identify a cap site using a mouse totalbrain cDNA library prepared by an oligo-cap method (Reference 32,Reference 33).

Since an adapter consisting of the nucleotide sequence represented bySEQ ID NO:21 is attached to the cap site, a primer consisting of thenucleotide sequence represented by SEQ ID NO:22 (a sequence consistingof nucleotides at positions 1 to 21 in the nucleotide sequencerepresented by SEQ ID NO:21) and a primer consisting of the nucleotidesequence represented by SEQ ID NO:23 (a sequence consisting ofnucleotides at positions 11 to 30 in the nucleotide sequence representedby SEQ ID NO:21) were prepared for the adapter. For the mouse Period2gene, on the other hand, a primer consisting of the nucleotide sequencerepresented by SEQ ID NO:24 [a sequence complimentary to a sequenceconsisting of nucleotides at positions 439 to 459 in the mouse Period2gene cDNA (GenBank AFO36893)], a primer consisting the nucleotidesequence represented by SEQ ID NO:25 [a sequence complimentary to asequence consisting of nucleotides at positions 377 to 398 in the mousePeriod2 gene cDNA (GenBank AFO036893)] and a primer consisting of thenucleotide sequence represented by SEQ ID NO:26 [a sequencecomplimentary to a sequence consisting of nucleotides at positions 300to 319 in the mouse Period2 gene cDNA (GenBank AFO36893)] were prepared.

When a nested PCR containing the first PCR which used the primerconsisting of the nucleotide sequence represented by SEQ ID NO:22 andthe primer consisting of the nucleotide sequence represented by SEQ IDNO:24 and the second PCR which used the primer consisting of thenucleotide sequence represented by SEQ ID NO:23 and the primerconsisting of the nucleotide sequence represented by SEQ ID NO:25 wascarried out, a DNA fragment of 414 bp containing the cap site wasobtained. Also, when another nested PCR containing the first PCR whichused the primer consisting of the nucleotide sequence represented by SEQID NO:22 and the primer consisting of the nucleotide sequencerepresented by SEQ ID NO:24 and the second PCR which used the primerconsisting of the nucleotide sequence represented by SEQ ID NO:23 andthe primer consisting of the nucleotide sequence represented by SEQ IDNO:26 was carried out, a DNA fragment of 335 bp containing the cap sitewas obtained.

As a result of the sequence analysis of these DNA fragments, it wasfound that the transcription start site is present in 8,440 bp upstream(nucleotide at position 7,736 in the nucleotide sequence represented bySEQ ID NO:1) from the translation start site (nucleotide at position16,176 in the nucleotide sequence represented by SEQ ID NO:1). Based onthis, it was confirmed that the transcription start site is contained inthe constructed vector pCH1 and vector pCH3, the vector pCH1 hasnucleotides of −3,321 to +3,142 (when the transcription start site isdefined as “+1”) in the mouse Period2 gene, and the vector pCH3 hasnucleotides of −3,321 to +196 in the mouse Period2 gene.

Example 3

<Analysis of Promoter Activity and Enhancer Activity of the UpstreamRegion of Mouse Period2 Gene>

A mouse cultured cell line NIH3T3 was inoculated at 1×10⁵ cells per wellinto a 6 well plate on the day before a luciferase assay. UsingLipofectamine 2000 (GIBCO-BRL) as the transfection reagent and using 1μg of each of various reporter vectors and 20 ng of an internal controlvector (PRL-SV40; Promega), transfection was carried out in accordancewith the manufacture's instructions. As the reporter vectors, the vectorpCH1 and vector pCH3 obtained in the above Example 1, a vector pGL3-bhaving no promoter activity (Promega) as a negative control and a vectorpGL3-promoter in which the SV40 promoter is connected to the upstream ofluciferase gene (Promega; to be referred to as pGL3-p hereinafter) as apositive control were used.

Also, the luciferase gene contained in vectors other than the internalcontrol vector (PRL-SV40) (namely vector pGL3-b, vector pCH1, vectorpCH3 and vector pGL3-p) is a firefly origin, while the luciferase genecontained in the internal control vector (PRL-SV40) is a sea pansyorigin.

After a lapse of 48 hours from the transfection, the cells were washedonce with phosphate buffered saline (PBS) and then the assay was carriedout using an assay kit (Picka Gene Dual Reporter Assay Kit; Nippon Gene)in accordance with the manufacture's instructions. The luminescencemeasurement was carried out using Microtiter Luminometer (DynatechLaboratories).

The results are shown in FIG. 3. In FIG. 3, “BLK” means an amount ofluminescence in the host cell not treated with transfection. Also, the“luciferase relative activity (normalized)” shown in FIG. 3 means avalue normalized based on the expression level of luciferase originatedfrom the internal control vector (PRL-SV40). As shown in FIG. 3, basalpromoter activity of the vector pCH1 was a low activity (7% of thevector pGL3-p), while the vector pCH3 showed a promoter activity of 55%of the vector pGL3-p.

In order to evaluate whether or not the vector pCH3 which contains thefirst exon upstream three conserved segmentss (I, II and III) among theconserved seven sevens (from I to VII) is functional, whether or not thereporter activity of pCH3 is enhanced by a BMAL1/CLOCK heterodimer,which act as a trans-acting factor for Period, was examined.

That is, transfection was carried out using Lipofectamine 2000(GIBCO-BRL) by adding 25 ng, 50 ng or 250 ng of each of pCl-neo-Bmal1and pCl-neo-Clock, together with the vector pCH3 (10 ng). Also, thevector pCl-neo-Bmal1 and vector pCl-neo-Clock have been prepared byintroducing mouse Email or mouse Clock, respectively, into a pCl-neovector (Promega) (Reference 34), and the mouse Bmal1 and mouse Clock areexpressed constitutionally in cultured cells. Also, in carrying out theabove transfection, 0.5 ng of pRL-CMV (Promega) was added as an internalcontrol, and the DNA to be transfected was adjusted with the pCl-neovector (Promega) to a total amount of 1 μg.

After a lapse of 48 hours from the transfection, luminescence level wasmeasured using an assay kit (Pica Gene Dual Reporter Assay Kit; NipponGene) in the same manner as the above procedure.

The results are shown in FIG. 4. The “BLK” in FIG. 4 has the samemeaning as the “BLK” shown in FIG. 3. As shown in FIG. 4, dose-dependenttranscriptional activation by the BMAL1/CLOCK heterodimer was confirmed.Based on this result, it was found that the thus constructed luciferasevector containing the upstream region of mouse Period2 gene isfunctional.

Example 4

<Preparation of mPer2:luc Transgenic Rat>

Since it is considered, based on the results of in vitro experimentscarried out in the above Example 3, that the vector pCH3 containssufficient elements for showing an intrinsic activity of the Period2gene expression, an attempt was made to prepare an mPer2:luc transgenicrat using this.

Firstly, in preparing a gene-introduced rat, sequences originating fromthe vector itself (e.g., replication origin ori, ampicillin resistancegene, etc.) were removed by treatment with restriction enzymes, SalI andMluI, and subsequent agarose gel electrophoresis. Purification of thefragment of interest was carried out using QIAquick Gel Extraction Kits(QIAquick; QUIAGEN), and after carrying out its phenol/chloroformtreatment, a DNA solution (concentration=75 ng/μl) was prepared bydissolving it in 100 μl of sterile TE buffer [10 mmol/l Tris-HCl (pH8.0), 1 mmol/l EDTA (pH 8.0)]. The above DNA fragment contains asequence consisting of nucleotides at positions 4,415 to 7,931 in thenucleotide sequence represented by SEQ ID NO:1 and a gene encodingluciferase.

In order to prepare a transgenic rat, Wistar rats were purchased fromCharles River Japan. The transgenic rat preparation operation wascarried out basically based on a known method (Reference 35), andmicro-injection of the DNA solution for injecting into rat pronucleusstage fertilized eggs was carried out in the following manner.

That is, sexually matured Wistar rats of 8-weeks-old were reared underconditions of 12 hour light-dark cycle (from 4:00 to 16:00 was used asthe light period), 23±2° C. in temperature and 55±5% in humidity, andthe hormone treating day was selected by observing sexual cycle offemales by vaginal smear. Firstly, 150 IU/kg of a pregnant mare serumgonadotropic hormone [PMS Zen-yaku (pregnant mare serum gonadotropin;PMSG); Nippon Zen-yaku] was intraperitoneally administered to femalerats to carry out superovulation treatment, 75 IU/kg of a humanchorionic gonadotropic hormone [Puvelogen (human chorionic gonadotropin;hCG); Sankyo Zoki] was administered 48-hours thereafter, and thencrossing was carried out by allowing them to lodge with males. After alapse of 32 hours from the hCG administration, pronucleus stagefertilized eggs were collected by oviduct perfusion. The mKRB solution(Reference 36) was used for the oviduct perfusion and culturing of eggs.After removing cumulus cells by carrying out an enzyme treatment of thecollected fertilized eggs at 37° C. for 5 minutes in mKRB solutioncontaining 0.1% hyaluronidase (Hyaluronidase Type I-S; Sigma), the eggswere washed three times with mKRB solution to remove the enzyme and thenstored in a CO₂ incubator (5% CO₂, 37° C., saturation humidity) untilthe DNA injection operation. The DNA solution prepared in the above wasinjected into male pronuclei of the thus prepared rat fertilized eggs.The injection operation was carried out for 525 embryos, and amongsurvived 431 embryos, 420 morphologically normal embryos weretransplanted into oviducts of pseudopregnancy-induced allomothers.

In order to examine whether or not exogenous DNA (including luciferasegene) was introduced into the rats, a primer consisting of thenucleotide sequence represented by SEQ ID NO:27 [a sequence consistingof nucleotides at positions 410 to 432 in the cloning vector pGL3-b(GenBank U47295)] and a primer consisting of the nucleotide sequencerepresented by SEQ ID NO:28 [a sequence complimentary to a sequenceconsisting of nucleotides at positions 980 to 1,000 in the cloningvector pGL3-b (GenBank U47295)] were designed as PCR primers for theluciferase gene. Introduction of exogenous DNA (including luciferasegene) can be verified by the presence or absence of a DNA product (591bp) amplified by the PCR using these primers.

A total of 65 rats were born by the transplantation of 420 embryos. Atthe time of 3-weeks-old, about 1 cm of the tail tip of each bornindividual was cut out using a surgical knife and dissolved by adding800 μl of a lysis solution and shaking overnight in a 55° C. constanttemperature bath. In this case, the above lysis buffer was prepared bydissolving actinase E and protease K, both to a final concentration of10 mg/ml, in a lysis buffer [a solution containing, as finalconcentrations, 50 mmol/l Tris-HCl (pH 8.0), 100 mmol/l EDTA (pH 8.0),100 mmol/l NaCl and 1% SDS].

Subsequently, phenol treatment was carried out twice, and the upperwater layer after centrifugation was collected and transferred into atube containing isopropanol. After mixing, the thus formed filamentousgenomic DNA was wound with the tip of a processed glass micro-pipette,soaked in 70% ethanol for 5 minutes and then in 100% ethanol for 5minutes and finally dissolved by soaking in TE, thereby preparinggenomic DNA of each individual.

The PCR which used the genomic DNA of each individual as the templatewas carried out using a Taq DNA polymerase (Roche Diagnostics) byincubating at 94° C. for 1 minute and then repeating 40 times of a cycleconsisting of 94° C. (30 seconds), 57° C. (30 seconds) and 72° C. (60seconds). As a result, it was found that 8 of the 65 born rats weremPer2:Luc transgenic rats.

In addition, at the time of 7-weeks-old, about 5 mm of the tail tip ofeach of the 8 mPer2:Luc transgenic rats was cut out using a surgicalknife. A luciferin solution was applied to the cut surface, and, turningup the cut surface, the luminescence was measured using aphotomultiplier tube detector [Photomal (model name LM-300Y2); HamamatsuPhotonics]. In this case, the above luciferin solution was prepared byadding HEPES (pH 7.2; final concentration=10 mmol/l), penicillinantibiotic (final concentration=25 U/ml), streptomycin antibiotic (finalconcentration=25 μg/ml), sodium bicarbonate (NaHCO₃; finalconcentration=0.3 g/l) and luciferin [Cat. E1601 (Promega); finalconcentration=0.1 mmol/l] to a Phenol Red-free DMEM medium (cat.13000-054; GIBCO-BRL).

As a result, tails of 4 of the 8 rats showed 923 cps, 4,392 cps, 1,122cps and 865 cps, respectively. Since these luminescence levels weresignificantly high compared to the around 100 cps of the control (tailof wild type rat), it was confirmed that 4 animals of the mPer2:Luctransgenic rat in which the luciferase functions in the living body wereobtained.

Among these 4 female transgenic rats (FO), the line showing the highestlevel of luminescence from tail (4,392 cps) was crossed with a wild typemale, and 11 F1 rats (5 males and 6 females) were obtained.

Example 5

<Operation of Automatic Measurement of Luminescence Levels fromSuprachiasmatic Nucleus Section and Peripheral Tissues (Liver Section,Lung Section and Eyeball) of mPer2:luc Transgenic Rat>

Since the F1 rats obtained in Example 4 are cross-breeds of wild typeand hetero transgenic rat, wild type and hetero transgenic rats areincluded in their legitimate F1 children. In order to select transgenicrats to be subjected to the test in this Example from the above F1 rats,tails of the above F1 rats (namely, the F1 rats obtained by the crossingof the line having the most highest tail luminescence with a wild type,prepared in Example 4) were cut off at the time of 6-weeks-old, andwhether or not they show luminescence was examined in the same manner asin Example 4. As a result, it was found that F1 transgenic rats showingsignificant luminescence were 8 animals (4 males and 4 females). Each ofsuprachiasmatic nucleus section as a rhythm center and peripheraltissues (liver section, lung section and eyeball) was prepared from onefemale (7-week-old) among these 8 transgenic rats in accordance with thefollowing procedure, and real time oscillation measurement was carriedout.

Firstly, preparation of suprachiasmatic nucleus sections was carried outin the following manner. That is, the transgenic rat was anesthetizedunder diethyl ether and sacrificed by cervical dislocation, both eyeswere excised to block input by light and then excision of total brainwas carried out. Unnecessary temporal part, frontal lobe and cerebellumamong the total brain were excised, and the resulting brain was fixed onan ice-cooled slicer table with an adhesive (Alon Alfa 201; To a Gosei).The brain fixed on the slicer table was filled with Hanks buffer [1×Hanks Buffer (GIBCO-BRL; cat. 14060-057) and 10 mmol/l HEPES (pH 7.2)(GIBCO-BRL; cat. 15630-080) in final concentrations], and preparation ofsections (400 μm in thickness) was carried out using a slicer(Microslicer DTK-1000; Dohan E M) while ice-cooling. A sectioncontaining the suprachiasmatic nucleus was selected by observing under astereoscopic microscope (SMZ645; Nikon). Preparation of thesuprachiasmatic nucleus section was carried out by cutting out thesuprachiasmatic nucleus alone located under the third cerebral ventriclefrom this section using a surgical knife under the stereoscopicmicroscope.

On the other hand, 1.2 ml of a luciferin-containing measuring medium[prepared by adding 10 mmol/l HEPES (pH 7.2) (GIBCO-BRL; cat.15630-080), 0.1 mmol/l luciferin potassium salt (Promega), antibiotics(25 U/ml penicillin and 25 μg/ml streptomycin; GIBCO-BRL) and 0.3 g/lNaHCO₃ (Wako Pure Chemical Industries), to respective finalconcentrations, to Phenol Red-free DMEM (Dulbecco's modified Eagle'smedium; GIBCO-BRL; cat. 13000-054)] was added to a 35 mm dish, and amembrane filter (MiliCell CM; Millipore; cat. PICMORG50) was floated onthe medium. The suprachiasmatic nucleus section prepared in the abovewas put on this membrane filter to which the medium was supplied fromthe bottom, and a slide glass cover (40 mm×50 mm; Matsunami GlassIndustry) was put on the 35 mm dish and the space between them wassealed by applying a silicon grease compound (Toray Dow Corning) toprevent drying of the medium during the culture. Automatic measurementof luminescence level (real time monitoring) was carried out by settingthe 35 mm dish containing suprachiasmatic nucleus section prepared inthis manner on inside of an photomultiplier tube detector (LM-300Y2;Hamamatsu Photonics) connected to a computer. The automatic measurementof luminescence level was continued for 20 days, and the measuringmedium was exchanged only once during the period (on the 10th day aftercommencement of the measurement).

Regarding the peripheral tissues, each of the liver, lung and eyeballwas excised from the transgenic rat. Thereafter, regarding the liver andlung, tissue sections of about 1 mm square were prepared using asurgical knife and the following measurement was carried out. Regardingthe eyeball on the other hand, the following measurement was carried outusing the excised tissue as such. A medium was prepared by adding agrowth enhancing agent B-27 additive (50×) [B-27 Supplement (50×);GIBCO-BRL] to a final concentration of 2% (1×) to the same aboveluciferin-containing measuring medium used for the culturing ofsuprachiasmatic nucleus section, each peripheral tissue (liver section,lung section or eyeball) was directly put into the medium and sealedwith a cover glass, and then automatic luminescence measurement wascarried out using the photomultiplier tube detector in the same manneras the case of the suprachiasmatic nucleus section. The automaticluminescence measurement was continued for 20 days, and the measuringmedium was exchanged only once during the period (on the 10th day aftercommencement of the measurement).

Results of the automatic luminescence measurement of the suprachiasmaticnucleus section during the first 10 days (namely from the commencementof the measurement until the lapse of 10 days) are shown in FIG. 5 andFIG. 6, and results of the automatic luminescence measurement of theperipheral tissues (liver section, lung section and eyeball) during thefirst 10 days are shown in FIG. 7, FIG. 8 and FIG. 9, respectively. InFIG. 5 to FIG. 9, the abscissa shows the number of days from thecommencement of the measurement (the measurement starting day wasexpressed as day 0), and the ordinate shows luminescence (cpm). Also,FIG. 6 shows a part of the graph shown in FIG. 5 expanded only invertical direction.

As shown in FIG. 5 and FIG. 6, luminescence quantity of thesuprachiasmatic nucleus section showed an oscillation peak after about 9hours on the 3rd day from the commencement of the measurement, and theoscillation rhythm was found thereafter at intervals of about 24 hours.Also, this oscillation continued until on the 20th day after thecommencement of the measurement.

As shown in FIG. 7, luminescence quantity of the liver section showedoscillation peaks after about 18 hours on the 3rd day and after about 18hours on the 4th day from the commencement of the measurement, but theoscillation was not observed thereafter due to its attenuation. Inaddition, since the oscillation rhythm recovered to 4 times by themedium exchange on the 10th day after commencement of the measurement,it is considered that the attenuation of oscillation rhythm is not dueto death of cells but due to de-synchronization among cells.

As shown in FIG. 8, the luminescence quantity of the lung section showeda total of 5 oscillation rhythms after the commencement of themeasurement, though accompanied by rapid changes.

As shown in FIG. 9, the luminescence quantity of the eyeball showed anoscillation rhythm having a peak after about 14 hours on the 1st day ofthe commencement of the measurement, and the oscillation rhythm wasobserved three times thereafter though the oscillation was small.

Thus, though there are differences in the oscillation phase andcontinuing frequency of oscillation among respective tissues of thesuprachiasmatic nucleus and peripheral tissues (liver, lung andeyeball), the oscillation rhythm after about 24 hours was observed inall of the tissues. It is considered that screening of a substancecapable of controlling expression of the Period2 gene becomes possibleby the automatic Period2 gene expression monitoring system which usesthe transgenic rat of the present invention or its suprachiasmaticnucleus sections or peripheral tissues.

Example 6

<Confirmation of Oscillation-Inducing Ability of Mouse Period2 Promoterin Cultured Cell Line>

It has been reported that when a rat cultured cell Rat-1 is stimulatedwith high concentration horse serum or DEX, a group of cells aresynchronized, and clock genes and a group of genes controlled by theclock genes start the 24 hour interval expression oscillation all atonce, which continues for several days and then attenuates (Reference2).

An attempt was made to introduce the above pCH3 construct prepared byconnecting a mouse Period2 gene upstream region to a luciferase vector,transiently into the Rat-1 cell, to carry out DEX stimulationthereafter, to change the medium to a medium containing luciferase andthen to measure luminescence therefrom real time using the aboveultra-weak emission counter. Its illustrative method is described below.Firstly, the Rat-1 cell (purchased from ATCC: designation=Rat1−R12; ATCCnumber=CRL-2210) was cultured and maintained at 37° C. under anatmosphere of 5% CO₂ in a 225 cm² flask (Iwaki) charged with 2 ml of amedium (prepared by adding antibiotics in respective finalconcentrations (100 U/ml penicillin and 100 μg/ml streptomycin;GIBCO-BRL) to Phenol Red-containing DMEM (cat. 11965-092)] containing10% FBS (fetal bovine serum; JRH Bioscience). The Rat-1 cell wasinoculated at 1.2×10⁶ cells per well into a 35 mm dish (Falcon) on theday before the transfection, and the culturing was continued using 2 mlof the above medium containing 5% FBS. On the next day, 1 μg of the pCH3construct was subjected to transfection using a transfection reagentLipofectamine 2000 (GIBCO-BRL) in accordance with the manufacture'sinstructions attached thereto. Three hours after the transfection, themedium was exchanged with 2 ml of fresh above medium containing 10% FBSto continue the culturing. Sixteen hours after the transfection, thestimulation was started by changing the medium to the above medium(containing 10% FBS) containing 0.1 mmol/l at final concentration of DEX(SIGMA; cat. D-8893). After 2 hours of the DEX stimulation, the mediumwas exchanged with 2 ml of a luciferin-containing measuring medium (theabove medium (containing 10% FBS) containing 0.1 mmol/l at finalconcentration of luciferin potassium salt (Promega)). After addition ofthe luciferin-containing measuring medium, this was covered with a slideglass, sealed using a silicon grease compound and arranged in theultra-weak emission counter connected to a computer, and then automaticluminescence measurement (real time monitoring) was carried out.

The results are shown in FIG. 10. The abscissa in FIG. 10 shows thenumber of days from the commencement of the measurement (the measurementstarting day was expressed as day 0), and the ordinate showsluminescence quantity (cpm). As shown in FIG. 10, oscillation of theluminescence was observed at intervals of 24 hours. Based on this, itwas confirmed that the mouse Period2 gene upstream region claimed by us(positions 4,415 to 7,931 in the nucleotide sequence represented by SEQID NO:1) has the oscillating ability also in cultured cells.Accordingly, it is considered that screening of a substance capable ofchanging the oscillation in cultured cells is possible by the use ofthis region.

Example 7

<Construction of Construct pTM15 in Which Human Period2 UpstreamSequence is Connected to Luciferase Vector>

From the results of mouse promoter activity analyses of Example 3 andExample 5, it was suggested that the regions I to III among the sevenconserved segments are considered to be sufficient for the oscillation.Accordingly, in order to obtain a DNA fragment containing human I to IIIregions (nucleotides at positions 3,820 to 6,068 in the nucleotidesequence represented by SEQ ID NO:2), PCR was carried out using a humangenomic DNA (Clontech) as the template and using a Taq polymerase(Advantage-GC Genomic polymerase Mix; Clontech) as the enzyme. Using aforward primer consisting of the nucleotide sequence represented by SEQID NO:29 (a sequence consisting of nucleotides at positions 3,644 to3,671 in the nucleotide sequence represented by SEQ ID NO:2) and areverse primer consisting of the nucleotide sequence represented by SEQID NO:30-(a sequence complimentary to a sequence consisting ofnucleotides at positions 6,402 to 6,429 in the nucleotide sequencerepresented by SEQ ID NO:2), the PCR was carried out by incubating at94° C. for 1 minute, repeating 35 times of a cycle consisting of 94° C.(15 seconds), 58° C. (30 seconds) and 68° C. (4 minutes) and thenfinally incubating at 68° C. for 10 minutes. As a result of the PCR, aDNA fragment of 2.8 kbp was obtained. In general, nucleotide A is addedto the 3′-terminal of a PCR fragment amplified by Taq polymerase, sothat, in order to carry out efficient cloning of a DNA fragment to whichA is added, an attempt was made to prepare a cloning vector in whichnucleotide T is added to the SmaI site of the luciferase vectorpGL3-basic. That is, the pGL3-basic was digested by treating it with therestriction enzyme SmaI and then subjected to 1% agarose gelelectrophoresis, and the digested vector was extracted from the gel,added to a PCR solution containing Taq polymerase (Roche Diagnostic) andincubated at 70° C. for 2 hours for addition of oligonucleotide T to theSmaI cut site. The pGL3-basic TA vector prepared in this manner canclone a PCR fragment efficiently into the SmaI site. A reporter vectorpTM15 was prepared by inserting the 2.8 kbp PCR fragment obtained by theabove PCR into the pGL3-basic TA vector. The sequence of the upstreamregion moiety of the human Period2 gene in the vector pTM15 correspondsto nucleotides at positions 3,644 to 6,429 in the nucleotide sequencerepresented by SEQ ID NO:2 and contains the conserved segments I to III(positions 3,820 to 6,068 in the sequence represented by SEQ ID NO:2).

Example 8

<Identification of Human Period2 Transcription Start Site>

Although it was found in Example 2 that the transcription start site ofmouse Period2 is present inside the conserved segments I to III, thereis no information regarding the transcription start site of humanPeriod2. Accordingly, in order to identify the transcription start siteof human Period2, an attempt was made to identify the cap region using ahuman cerebellum cDNA library prepared by oligo-cap method (total RNA tobe used as the source was obtained from Clontech (cat. 64035-1);Reference 32 and Reference 33). That is, when a nested PCR containingthe first PCR which used a primer consisting of the nucleotide sequencerepresented by SEQ ID NO:22 and a primer consisting of the nucleotidesequence represented by SEQ ID NO:31 [a sequence complimentary to asequence consisting of nucleotides at positions 480 to 501 in a humanPeriod2 gene cDNA (GenBank NM-003894.1)] and the second PCR which used aprimer consisting of the nucleotide sequence represented by SEQ ID NO:23and a primer consisting of the nucleotide sequence represented by SEQ IDNO:32 [a sequence complimentary to a sequence consisting of nucleotidesat positions 122 to 143 in the human Period2 gene cDNA (GenBankNM-003894.1)] was carried out, two DNA fragments of 209 bp and 323 bpcontaining the cap region were obtained. As a result of the sequenceanalysis of these two DNA fragments, it was found that the first exon ofthe Period2 gene exists in two kinds (to be called exon 1A and exon 1Bfor convenience) which independently connect to the second exon(nucleotides at positions 16,462 to 16,710 in the nucleotide sequencerepresented by SEQ ID NO:2). The exon 1A is positions 5,625 to 5,773 inthe nucleotide sequence represented by SEQ ID NO:2, and the exon 1B ispositions 5,806 to 6,068 in the nucleotide sequence represented by SEQID NO:2. Thus, it was found that there are two human Period2 genetranscription start sites, and they are present at positions 5,625 (exon1A) and 5,806 (exon 1B) in the nucleotide sequence represented by SEQ IDNO:2. Based on this, it was confirmed that transcription start sites arecontained in the thus constructed vector pTM15.

Example 9

<Confirmation of Oscillation-Inducing Ability of Human Period2 Promoterin Cultured Cell Line>

As a result of examination on the oscillation-inducing ability of thehuman Period2 promoter vector pTM15 in cultured cell by the same methodof Example 6, significant oscillation shown in FIG. 11 was confirmed.The abscissa in FIG. 11 shows the number of days from the commencementof the measurement (the measurement starting day was expressed as day0), and the ordinate shows luminescence quantity (cpm). Since DNAfragment containing the human Period2 gene upstream region claimed by us(positions 3,820 to 6,068 in the nucleotide sequence represented by SEQID NO:2) showed the oscillating ability also in cultured cells, it isconsidered that screening of a substance capable of changing theoscillation in cultured cells is possible by the use of this region.

Example 10

<Preparation of Deletion Construct in Which Mouse Period2 UpstreamSequence is Connected to Luciferase Vector>

It was revealed that the vector pCH3 obtained in Example 1 (contains asequence of nucleotides at positions 4,415 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1), containing a mouse Period2 geneupstream region fragment having three conserved segments (I, II and III)of upstream of the first exon, has strong promoter activity as shown inFIG. 3 of Example 3 and is transcription-activated by a Periodtranscription activation factor BMAL1/CLOCK heterodimer as shown in FIG.4. In order to find that a region important for the basal promoteractivity of the mouse Period2 gene and a region important for thetranscriptional activation by BMAL1/CLOCK heterodimer are present inwhich of the three conserved segments, conserved segment deletionconstructs were prepared and examined. Firstly, a deletion constructconsisting of a shorter nucleotide sequence containing all of theconserved segments I, II and III (correspond to sequences consisting ofnucleotides at positions 5,932 to 6,043, 6,087 to 6,179 and 7,518 to7,735, respectively, in the nucleotide sequence represented by SEQ IDNO:1) was prepared. The above vector pCH3 was subjected to a treatmentwith restriction enzymes, MulI and BalI, and the digested DNA fragmentwas removed by an agarose gel electrophoresis. A vector pCH3-D1 wasprepared from the vector pCH3 by self-ligation of the thus deletion DNAfragment-deleted product. In the vector pCH3-D1, the sequence of theupstream region moiety of the mouse Period2 gene corresponds to asequence consisting of nucleotides at positions 5,249 to 7,931 in thenucleotide sequence represented by SEQ ID NO:1 (a nucleotide sequence of−2,487 to +196 when the transcription start site is defined as “+1”).Next, a deletion construct in which the preserved regions I and II weredeleted was prepared. The vector pCH3 was subjected to a treatment withrestriction enzymes, MulI and EcoRI, and the digested DNA fragment wasremoved by an agarose gel electrophoresis. A vector pCH3-D2 was preparedfrom the vector pCH3 by carrying out self ligation of the thus deletionDNA fragment-deleted product. In the vector pCH3-D2, the sequence of theupstream region moiety of the mouse Period2 gene corresponds to asequence consisting of nucleotides at positions 6,417 to 7,931 in thenucleotide sequence represented by SEQ ID NO:1 (a nucleotide sequence of−1,319 to +196 when the transcription start site is defined as “+1”). Inaddition, in order to construct a vector mainly containing the conservedsegment III alone, the vector pCH3 was subjected to a treatment withMulI/BstXI restriction enzymes, MulI and BstXI, and the digested DNAfragment was removed by an agarose gel electrophoresis. A vector pCH3-D3was prepared from the vector pCH3 by carrying out self-ligation of thethus deletion DNA fragment-deleted product. In the vector pCH3-D3, thesequence of the upstream region moiety of the mouse Period2 genecorresponds to a sequence consisting of nucleotides at positions 7,463to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 (anucleotide sequence of −273 to +196 when the transcription start site isdefined as “+1”).

Example 11

<Analysis of Promoter Activity and Enhancer Activity of DeletionConstructs of Mouse Period2 Upstream Region>

A mouse cultured cell line NIH3T3 was inoculated at 1×10⁵ cells per wellinto a 6 well plate on the day before a luciferase assay. UsingLipofectamine 2000 (GIBCO-BRL) as the transfection reagent and using 1μg of each of various reporter vectors and 20 ng of an internal controlvector (PRL-SV40; Promega), transfection was carried out in accordancewith the manufacture's instructions. As the reporter vectors describedabove, the vector pCH3-D1, vector pCH3-D2 and vector pCH3-D3 obtained inthe above Example 10, a vector pGL3-b having no promoter activity(Promega) as a negative control and the vector pGL3 as a positivecontrol were used.

Also, the luciferase gene contained in vectors other than the internalcontrol vector (PRL-SV40) (namely vector pCH3-D1, vector pCH3-D2, vectorpCH3-D3 and vector pCH3) is a firefly origin, while the luciferase genecontained in the internal control vector (PRL-SV40) is a sea pansyorigin.

Forty-eight hours after the transfection, the cells were washed oncewith phosphate buffered saline (PBS) and then the assay was carried outusing an assay kit (Pica Gene Dual Reporter Assay Kit; Nippon Gene) inaccordance with the manufacture's instructions. The luminescence wasmeasured using Microtiter Luminometer (Dynatech Laboratories).

The results are shown in FIG. 12. In FIG. 12, “BLK” means an amount ofluminescence in the host cell not treated with transfection. Also, the“luciferase relative activity (standardized)” shown in FIG. 12 means avalue standardized based on the luciferase expression quantityoriginated from the internal control vector (PRL-SV40). As shown in FIG.12, the basal promoter activity of the vector pCH3-D1 and vector pCH3-D2showed a promoter activity of about 60% of the vector pCH3. The basalpromoter activity of the vector pCH3-D3 showed a promoter activity ofabout 80% of the vector pCH3. Based on the above, it was suggested thatthe region carrying out basal promoter activity of the mouse Period ispresent in a sequence consisting of nucleotides at positions 7,463 to7,931 in the nucleotide sequence represented by SEQ ID NO:1. When thefact that the transcription start site and the conserved segment III arecontained in this region is taken into consideration, it is consideredthat the conserved segment III is important for the basal promoteractivity.

Next, a region necessary for the Period2 to undergo transcriptionalactivation by the trans-acting factor BMAL1/CLOCK was examined.

Transfection was carried out using Lipofectamine 2000 (GIBCO-BRL) byadding 250 ng of each of pCI-neo-Bmal1 and pCI-neo-Clock together with10 ng of the above vector pCH3-D1, vector pCH3-D2, vector pCH3-D3 orvector pCH3. In carrying out the transfection, 0.5 ng of pRL-CMV(Promega) was added as an internal control, and the DNA to betransfected was adjusted with the pCl-neo vector (Promega) to a totalamount of 1 μg.

Forty-eight hours after the transfection, the measurement ofluminescence level was carried out using an assay kit (Pica Gene DualReporter Assay Kit; Nippon Gene) in the same manner as in the aboveprocedure.

The results are shown in FIG. 12. The “BLK” in FIG. 12 has the samemeaning as the “BLK” shown in FIG. 4. As shown in FIG. 12,transcriptional activity of all of the vector pCH3-D1, vector pCH3-D2,vector pCH3-D3 and vector pCH3 was enhanced 5.0 times, 5.6 times, 7.1times and 5.6 times, respectively, by the co-transfection of Bmal1 geneand Clock gene, so that it was considered that they receivedtranscriptional activation by the BMAL1/CLOCK heterodimer. Based on thisresult, it was suggested that the sequence important for the mousePeriod2 to undergo transcription activation by the transcriptionactivation factor BMAL1/CLOCK heterodimer is present in a sequenceconsisting of nucleotides at positions 7,463 to 7,931 in the nucleotidesequence represented by SEQ ID NO:1. Since the conserved segment III iscontained in this region, it is considered that a responsive element, onwhich the BMAL1/CLOCK heterodimer functions, is present in the conservedsegment III.

Example 12

<Analysis of Oscillation-Inducing Ability of Mouse Period2 UpstreamRegion Deletion Construct>

As a result of the measurement of oscillation-inducing ability incultured cells using the vector pCH3-D3 prepared in Example 10, carriedout by a method similar to that in Example 6, significant oscillationshown in FIG. 13 was confirmed. The abscissa in FIG. 13 shows the numberof days from the commencement of the measurement (the measurementstarting day was expressed as day 0), and the ordinate showsluminescence quantity (cpm). Since a DNA fragment containing only theconserved segment III (corresponds to a sequence consisting ofnucleotides at positions 7,463 to 7,931 in the nucleotide sequencerepresented by SEQ ID NO:1) among the mouse Period2 gene upstream regionshowed the oscillating ability in cultured cells, it is considered thata sequence important for the oscillatory expression is present in theconserved segment III of mouse Period2 and that screening of a substancecapable of changing the oscillatory expression in cultured cells ispossible by the use of this region.

INDUSTRIAL APPLICABILITY

A system for the screening of substances capable of controllingexpression of biological clock genes can be constructed by using thePeriod2 gene promoter of the present invention, the construct of thepresent invention containing this promoter and a reporter gene, the cellof the present invention, the transgenic animal of the present inventionor its suprachiasmatic nucleus sections or peripheral tissues.Substances selected by this screening system are useful as candidatesubstances of agents for improving circadian rhythm disorders (e.g.,sleep disturbance, depression or abnormal behavior of patients withsenile dementia, caused by abnormal circadian rhythm).

LIST OF REFERENCES

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Explanation of “Artificial Sequence” is described in the numerical entry<223> in the following Sequence Listing. Specifically, the nucleotidesequence represented by the sequence of SEQ ID NO:21 in the SequenceListing is an adapter sequence of oligo cap. The nucleotide sequencerepresented by the sequence of SEQ ID NO:22 in the Sequence Listing is asequence consisting of nucleotides at positions 1 to 21 in thenucleotide sequence represented by the sequence of SEQ ID NO:21 in theSequence Listing. The nucleotide sequence represented by the sequence ofSEQ ID NO:23 in the Sequence Listing is a sequence consisting ofnucleotides at positions 11 to 30 in the nucleotide sequence representedby the sequence of SEQ ID NO:21 in the Sequence Listing. The nucleotidesequence represented by the sequence of SEQ ID NO:27 in the SequenceListing is a sequence consisting of nucleotides at positions 410 to 432in the cloning vector pGL3-b (GenBank U47295). The nucleotide sequencerepresented by the sequence of SEQ ID NO:28 in the Sequence Listing is asequence complimentary to a sequence consisting of nucleotides atpositions 980 to 1,000 in the cloning vector pGL3-b (GenBank U47295).

Although the invention has been described in the above based onspecified embodiments, modifications and improvements obvious to thoseskilled in the art are included in the scope of the present invention.

1-10. (canceled)
 11. An isolated DNA which maintains a basal promoteractivity and has a promoter activity transcriptionally-activated by aBMAL1/CLOCK heterodimer, which comprises the nucleotide sequenceconsisting of nucleotides at positions 3,820 to 6,068 in the nucleotidesequence represented by SEQ ID NO:2.
 12. The DNA according to claim 11,which consists of the nucleotide sequence of nucleotides at positions3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2.13. A construct which comprises the DNA according to claim 11 or 12operably linked to a reporter gene.
 14. A cell which comprises theconstruct according to claim
 13. 15. A method for screening a substancewhich controls expression of Period2 gene, comprising the steps of:allowing the cell according to claim 14 to contact with a substance tobe tested, and measuring activity of the reporter gene.
 16. A transgenicrat or mouse transfected with the construct according to claim 13, andwherein the suprachiasmatic nucleus and/or peripheral tissues of the rator mouse exhibit the function of reporter gene expression.
 17. Thetransgenic rat according to claim
 16. 18. The transgenic rat or mouseaccording to claim 16, wherein the reporter gene is one member selectedfrom the group consisting of a gene encoding luciferase, a gene encodingsecretion type alkaline phosphatase (SEAP), a gene encoding greenfluorescent protein (GFP), a gene encoding chloramphenicalacetyltransferase (CAT), a gene encoding β-glucuronidase (GUS), a geneencoding β-D-galactosidase and a gene encoding aequorin.
 19. Thetransgenic rat according to claim 17, wherein the reporter gene is onemember selected from the group consisting of a gene encoding luciferase,a gene encoding secretion type alkaline phosphatase (SEAP), a geneencoding green fluorescent protein (GFP), a gene encodingchloramphenical acetyltransferase (CAT), a gene encoding β-glucuronidase(GUS), a gene encoding β-D-galactosidase and a gene encoding aequorin.20. A method for screening a substance which controls expression and/oroscillatory expression of Period2 gene, comprising the steps of:allowing the cell according to claim 14 to react with a substance to betested, and measuring activity of the reporter gene for oscillatoryexpression of the Period2 gene.
 21. A method for screening a substancewhich controls expression and/or oscillatory expression of Period2 gene,comprising the steps of: administering a substance to be tested to thetransgenic rat or mouse according to claim 16, and measuring activity ofthe reporter gene in the suprachiasmatic nucleus of the animal foroscillatory expression of the Period2 gene.
 22. A method for screening asubstance which controls expression and/or oscillatory expression ofPeriod2 gene, comprising the steps of: allowing a suprachiasmaticnucleus section or peripheral tissue of the transgenic rat or mouseaccording to claim 16 to react with a substance to be tested, andmeasuring activity of the reporter gene for oscillatory expression ofthe Period2 gene.